U.S. patent number 9,533,059 [Application Number 13/816,589] was granted by the patent office on 2017-01-03 for peptide radiotracer compositions.
This patent grant is currently assigned to GE HEALTHCARE LIMITED. The grantee listed for this patent is Rajiv Bhalla, Gareth Getvoldsen, Bard Indrevall, Peter Brian Iveson. Invention is credited to Rajiv Bhalla, Gareth Getvoldsen, Bard Indrevall, Peter Brian Iveson.
United States Patent |
9,533,059 |
Iveson , et al. |
January 3, 2017 |
Peptide radiotracer compositions
Abstract
The present invention relates to imaging agent compositions
comprising radiolabelled c-Met binding peptides suitable for
positron emission tomography (PET) imaging in vivo. The c-Met
binding peptides are labelled with the radioisotope .sup.18F. Also
disclosed are pharmaceutical compositions, methods of preparation
of the agents and compositions, plus methods of in vivo imaging
using the compositions, especially for use in the management of
cancer.
Inventors: |
Iveson; Peter Brian
(Buckinghamshire, GB), Bhalla; Rajiv
(Buckinghamshire, GB), Indrevall; Bard (Oslo,
NO), Getvoldsen; Gareth (Buckinghamshire,
GB) |
Applicant: |
Name |
City |
State |
Country |
Type |
Iveson; Peter Brian
Bhalla; Rajiv
Indrevall; Bard
Getvoldsen; Gareth |
Buckinghamshire
Buckinghamshire
Oslo
Buckinghamshire |
N/A
N/A
N/A
N/A |
GB
GB
NO
GB |
|
|
Assignee: |
GE HEALTHCARE LIMITED
(Buckinghampshire, GB)
|
Family
ID: |
42938098 |
Appl.
No.: |
13/816,589 |
Filed: |
August 11, 2011 |
PCT
Filed: |
August 11, 2011 |
PCT No.: |
PCT/EP2011/063890 |
371(c)(1),(2),(4) Date: |
February 12, 2013 |
PCT
Pub. No.: |
WO2012/022676 |
PCT
Pub. Date: |
February 23, 2012 |
Prior Publication Data
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|
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Document
Identifier |
Publication Date |
|
US 20130149241 A1 |
Jun 13, 2013 |
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Foreign Application Priority Data
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Aug 18, 2010 [GB] |
|
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1013808.9 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K
7/64 (20130101); A61K 49/00 (20130101); A61K
51/088 (20130101); A61K 38/16 (20130101); A61K
38/1833 (20130101) |
Current International
Class: |
A61K
51/08 (20060101); C07K 7/64 (20060101); A61K
49/00 (20060101); A61K 38/18 (20060101); A61K
38/16 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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2009-542689 |
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Dec 2009 |
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JP |
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2010520229 |
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Jun 2010 |
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JP |
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2010526859 |
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Aug 2010 |
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JP |
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5309141 |
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Oct 2013 |
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JP |
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5341757 |
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Nov 2013 |
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JP |
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2004/078778 |
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Sep 2004 |
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WO |
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2004/080492 |
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Sep 2004 |
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WO |
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2006/030291 |
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Mar 2006 |
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WO |
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WO 2006/030291 |
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Mar 2006 |
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WO |
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2008/072976 |
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Jun 2008 |
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WO |
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2008/139207 |
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Nov 2008 |
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WO |
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WO 2008/139207 |
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Nov 2008 |
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WO |
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2009/016180 |
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Feb 2009 |
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WO |
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2009016181 |
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Feb 2009 |
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WO |
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WO 2009/016180 |
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Feb 2009 |
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WO |
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WO20090161180 |
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Feb 2009 |
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WO |
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WO2009-027706 |
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Mar 2009 |
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WO |
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WO 2009106566 |
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Sep 2009 |
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WO |
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Other References
Kilbourn et al., "Fluorine-18 Labeling of Proteins", J Nucl Med,
1987, pp. 462-470. cited by examiner .
Poethko et al., "Two-Step Methodology for High-Yield Routine
Radiohalogenation of Peptides: 18F-Labeled RGD and Octreotide
Analogs", J Nucl Med, 2004, pp. 892-902. cited by examiner .
Poethko Journal of Nuclear Medicine, Society of Nuclear Medicine,
Reston, VA, vol. 45, No. 5, May 1, 2004, pp. 892-902 "Two-Step
Methodology for High-Yield Routine Radiohalogenation of Peptide:
18F-Labled RGD and Octreotide Analogs". cited by applicant .
PCT/EP2011/063890 ISRWO Dated Dec. 19, 2011. cited by applicant
.
Great Britain 1013808.9 Search Report Dated Dec. 15, 2010. cited by
applicant .
Flavell Journal American Chemical Society, 2008, vol. 1330, pp.
9106-9112 "Site-Specific 18F-Labeling of the Protein Hormone Leptin
Using a General Two-Step Ligation Procedure". cited by applicant
.
Office Action (translation) issued in Chile Application No.
483-2013 (Dec. 17, 2015). cited by applicant .
Japanese Office Action issued May 17, 2016 in corresponding JP
Appl. No. 2013-524419. (English Translation attached). cited by
applicant.
|
Primary Examiner: Garyu; Lianko
Attorney, Agent or Firm: Wood IP LLC
Claims
The invention claimed is:
1. An imaging agent composition which comprises (i) a
.sup.18F-radiolabelled c-Met binding cyclic peptide and (ii) an
unlabelled c-Met binding cyclic peptide; wherein the unlabelled
c-Met binding cyclic peptide has the same amino acid sequence as
the radiolabelled c-Met binding cyclic peptide; wherein the
unlabelled c-Met binding cyclic peptide is present in the
composition at no more than 50 times the molar amount of the
.sup.18F-labelled c-Met binding cyclic peptide; and wherein the
c-Met binding cyclic peptide is an 18 to 30-mer cyclic peptide of
Formula I: Z.sup.1-[cMBP]-Z.sup.2 (I) where cMBP is of Formula II:
-(A).sub.x-Q-(A').sub.y- (II); and Q is the amino acid sequence SEQ
ID NO: 1: TABLE-US-00013
-Cys.sup.a-X.sup.1-Cys.sup.c-X.sup.2-Gly-Pro-Pro-X.sup.3-Phe-Glu-Cys.sup.d-
-Trp-Cys.sup.b-Tyr-X.sup.4-X.sup.5-X.sup.6-;
wherein X.sup.1 is Asn, His, or Tyr; X.sup.2 is Gly, Ser, Thr or
Asn; X.sup.3 is Thr or Arg; X.sup.4 is Ala, Asp, Glu, Gly or Ser;
X.sup.5 is Ser or Thr; X.sup.6 is Asp or Glu; and Cys.sup.a-d are
each cysteine residues such that residues a and b as well as c and
d are cyclised to form two separate disulfide bonds; A and A' are
independently any amino acid other than Cys, with the proviso that
at least one of A and A' is present and is Lys; x and y are
independently integers of value 0 to 13, and are chosen such that
[x+y]=1 to 13; Z.sup.1 is attached to the N-terminus of cMBP, and
is H or M.sup.IG; Z.sup.2 is attached to the C-terminus of cMBP and
is OH, OB.sup.c, or M.sup.IG, where B.sup.c is a biocompatible
cation; each M.sup.IG is independently a metabolism inhibiting
group which is a biocompatible group which inhibits or suppresses
in vivo metabolism of the cMBP; and wherein the labelled cMBP is
labelled at the Lys residue of the A or A' groups with .sup.18F,
and wherein the unlabelled c-Met binding cyclic peptide excludes
the c-Met binding cyclic peptide labelled with .sup.19F, where said
.sup.19F labelled c-Met binding cyclic peptide and
.sup.18F-radiolabelled c-Met binding cyclic peptide differ only in
the isotopes of the fluorine atom.
2. The imaging agent composition of claim 1, where cMBP is of
Formula IIA: -(A).sub.x-Q-(A').sub.z-Lys- (IIA) wherein: z is an
integer of value 0 to 2 and [x+z]=0 to 12, and cMBP comprises only
one Lys residue.
3. The imaging agent composition of claim 1, wherein cMBP comprises
the amino acid sequence of either SEQ ID NO: 2 or SEQ ID NO: 3:
TABLE-US-00014 (SEQ ID NO: 2)
Ser-Cys.sup.a-X.sup.1-Cys.sup.c-X.sup.2-Gly-Pro-Pro-X.sup.3-Phe-Glu-Cys.su-
p.d-Trp-Cys.sup.b-Tyr-X.sup.4-X.sup.5-X.sup.6; (SEQ ID NO: 3)
Ala-Gly-Ser-Cys.sup.a-X.sup.1-Cys.sup.c-X.sup.2-Gly-Pro-Pro-X.sup.3-Phe-Gl-
u-Cys.sup.d-Trp-Cys.sup.b-Tyr- X.sup.4-X.sup.5-X.sup.6-Gly-Thr.
4. The imaging agent composition of claim 1, wherein X.sup.3 is
Arg.
5. The imaging agent composition of claim 1, wherein either the
-(A).sub.x- or -(A').sub.y- groups comprise a linker peptide which
is chosen from: TABLE-US-00015 (SEQ ID NO: 4) -Gly-Gly-Gly-Lys-,
(SEQ ID NO: 5) -Gly-Ser-Gly-Lys- or (SEQ ID NO: 6)
-Gly-Ser-Gly-Ser-Lys-.
6. The imaging agent composition of claim 5, where cMBP has the
amino acid sequence (SEQ ID NO: 7): TABLE-US-00016
Ala-Gly-Ser-Cys.sup.a-Tyr-Cys.sup.c-Ser-Gly-Pro-Pro-Arg-Phe-Glu-Cys.sup.d--
Trp-Cys.sup.b-Tyr- Glu-Thr-Glu-Gly-Thr-Gly-Gly-Gly-Lys.
7. The imaging agent composition of claim 1, where both Z.sup.1 and
Z.sup.2 are independently M.sup.IG.
8. The imaging agent composition of claim 7, where Z.sup.1 is
acetyl and Z.sup.2 is a primary amide.
9. The imaging agent composition of claim 1, wherein cMBP is
labelled at epsilon amine group of the Lysine residue of the A or
A' groups with .sup.18F.
10. The imaging agent composition of claim 1, wherein cMBP is
labelled at epsilon amine group of the carboxy terminal Lysine with
.sup.18F.
11. The imaging agent composition of claim 1, further comprising
4-aminobenzoic acid as radioprotectant and 5-10% v/v ethanol as
solubilizer.
12. The imaging agent composition of claim 1, wherein the
.sup.18F-radiolabelled c-Met binding cyclic peptide has the
following structure: ##STR00009## wherein n=18 and Formula I is
radiolabelled at the Lysine residue of the A or A' groups of the
cMBP.
13. The imaging agent composition of claim 1, wherein the
.sup.18F-radiolabelled c-Met binding cyclic peptide has the
following structure: ##STR00010## wherein n=18 and Formula I is
radiolabelled at the Lysine residue of the A or A' groups of the
cMBP.
14. The imaging agent composition of claim 1, which is maintained
at or above pH 7.5 and/or further comprises a solubiliser.
15. The imaging agent composition of claim 1, which further
comprises one or more radioprotectants.
16. A pharmaceutical composition which comprises the imaging agent
composition of claim 1 together with a biocompatible carrier, in a
sterile form suitable for mammalian administration.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a filing under 35 U.S.C. 371 of international
application number PCT/EP2011/063890, filed Aug. 11, 2011, which
claims priority to Great Britain application number 1013808.9 filed
Aug. 18, 2010, the entire disclosure of which is hereby
incorporated by reference.
FIELD OF THE INVENTION
The present invention relates to imaging agent compositions
comprising radiolabelled c-Met binding peptides suitable for
positron emission tomography (PET) imaging in vivo. The c-Met
binding peptides are labelled with the radioisotope .sup.18F. Also
disclosed are pharmaceutical compositions, methods of preparation
of the agents and compositions, plus methods of in vivo imaging
using the compositions, especially for use in the diagnosis of
cancer.
BACKGROUND TO THE INVENTION
Hepatocyte growth factor (HGF), also known as scatter factor (SF),
is a growth factor which is involved in various physiological
processes, such as wound healing and angiogenesis. The high
affinity interaction of HGF interaction with its receptor (c-Met)
is implicated in tumour growth, invasion and metastasis.
Knudsen et al have reviewed the role of HGF and c-Met in prostate
cancer, with possible implications for imaging and therapy [Adv.
Cancer Res., 91, 31-67 (2004)]. Labelled anti-met antibodies for
diagnosis and therapy are described in WO 03/057155.
c-Met has been shown to be involved in tumour growth, invasion and
metastasis in many human cancers of epithelial origin. c-Met is
expressed by most carcinomas and its elevated expression relative
to normal tissue has been detected in cancers of: lung, breast,
colorectal, pancreatic, head and neck, gastric, hepatocellular,
ovarian, renal, glioma, melanoma and a number of sarcomas. In
colorectal carcinoma (CRC), over-expression of c-Met has been
detected in dysplastic aberrant crypt foci, the earliest
pre-neoplastic lesions of the disease. In head and neck squamous
cell cancer, c-Met is reportedly expressed or overexpressed in
roughly 80% of primary tumours. In prostate cancer metastasis to
bone, c-Met was reported overexpressed in over 80% of bone
metastasis.
Under normal conditions, c-Met is expressed on epithelial cells and
activated in a paracrine fashion, by mesenchymally derived HGF. The
activation of c-Met in normal cells is a transient event and is
tightly regulated. In tumour cells, however, c-Met can be
constitutively active. In cancer, aberrant c-Met stimulation can be
achieved through c-Met amplification/over-expression, activating
c-Met mutations (e.g. structural alterations) and acquisition of
autonomous growth control through creation of autocrine signalling
loops. In addition, a defective down-regulation of the c-Met
receptor will also contribute to aberrant c-Met expression in the
cell membrane. While the over-expression of c-Met is HGF dependent
(autocrine/paracrine), structural alterations caused by mutations
are HGF independent (e.g. loss of extracellular domain).
WO 2004/078778 discloses polypeptides or multimeric peptide
constructs which bind c-Met or a complex comprising c-Met and HGF.
Approximately 10 different structural classes of peptide are
described. WO 2004/078778 discloses that the peptides can be
labelled with a detectable label for in vitro and in vivo
applications, or with a drug for therapeutic applications. The
detectable label can be: an enzyme, a fluorescent compound, an
optical dye, a paramagnetic metal ion, an ultrasound contrast agent
or a radionuclide. Preferred labels of WO 2004/078778 are stated to
be radioactive or paramagnetic, and most preferably comprise a
metal which is chelated by a metal chelator. WO 2004/078778 states
that the radionuclides therein can be selected from: .sup.18F,
.sup.124I, .sup.125I, .sup.131I, .sup.123I, .sup.77Br, .sup.76Br,
.sup.99mTc, .sup.51Cr, .sup.67Ga, .sup.47Sc, .sup.167Tm,
.sup.141Ce, .sup.111In, .sup.168Yb, .sup.175Yb, .sup.140La,
.sup.90Y, .sup.88Y, .sup.153Sm, .sup.166Ho, .sup.165Dy, .sup.166Dy,
.sup.62Cu, .sup.64Cu, .sup.67Cu, .sup.97Ru, .sup.103Ru, .sup.186Re,
.sup.203Pb, .sup.211Bi, .sup.212Bi, .sup.213Bi, .sup.214Bi,
.sup.105Rb, .sup.109Pd, .sup.117mSu, .sup.149Pm, .sup.161Tb,
.sup.177Lu, .sup.198Au and .sup.199Au. WO 2004/078778 states (page
62) that the preferred radionuclides for diagnostic purposes are:
.sup.64Cu, .sup.67Ga, .sup.68Ga, .sup.99mTc and .sup.111In, with
.sup.99mTc being particularly preferred.
WO 2008/139207 discloses c-Met binding cyclic peptides of 17 to 30
amino acids which are labelled with an optical reporter imaging
moiety suitable for imaging the mammalian body in vivo using light
of green to near-infrared wavelength 600-1200 nm. The c-Met binding
peptides comprise the amino acid sequence SEQ ID NO: 1:
TABLE-US-00001
Cys.sup.a-X.sup.1-Cys.sup.c-X.sup.2-Gly-Pro-Pro-X.sup.3-Phe-Glu-Cys.sup.d--
Trp-Cys.sup.b-Tyr-X.sup.4-X.sup.5-X.sup.6;
wherein X.sup.1 is Asn, H is or Tyr; X.sup.2 is Gly, Ser, Thr or
Asn; X.sup.3 is Thr or Arg; X.sup.4 is Ala, Asp, Glu, Gly or Ser;
X.sup.5 is Ser or Thr; X.sup.6 is Asp or Glu; and Cys.sup.a-d are
each cysteine residues such that residues a and b as well as c and
d are cyclised to form two separate disulfide bonds. The optical
reporter of WO 2008/139207 is preferably a cyanine dye.
WO 2009/016180 discloses c-Met binding cyclic peptides analogous to
those of WO 2008/139207, wherein the optical reporter is a
benzopyrylium dye. The agents of WO 2008/139207 and WO 2009/016180
are stated to be useful for in vitro and in vivo optical
applications, especially optical imaging in vivo of the human body.
Optical imaging of colorectal cancer is a preferred
application.
SUMMARY OF THE INVENTION
The present invention relates to imaging agent compositions
comprising .sup.18F-radiolabelled c-Met binding peptides suitable
for positron emission tomography (PET) imaging in vivo. The c-Met
binding peptides are labelled via a lysine (Lys) residue.
The imaging agent compositions preferably suppress the level of
unlabelled c-Met binding cyclic peptide present. That is
advantageous because the .sup.18F-labelled cMBP is a radiotracer,
present at and administered in extremely low chemical
concentration--hence, if not removed, the unlabelled cMBP would
otherwise be present in large chemical excess. That has been
established to be important for PET in vivo imaging applications,
since otherwise the unlabelled cMBP competes effectively with the
.sup.18F-labelled cMBP for the c-Met binding sites in vivo. It thus
has a deleterious effect on the uptake and hence
signal-to-background ratio in vivo. This issue was not reported in
the prior art, since eg. when c-Met binding peptides are labelled
with optical reporter dyes, the chemical amounts of labelled
peptide involved are substantially greater than for PET, and hence
the competition issue does not arise.
The imaging agent compositions of the present invention also
overcome a previously unrecognised problem wherein, radiolabelled
cMBP peptides suffer adhesion problems to various materials,
including filters. Solubilised compositions are provided, which
means that .sup.18F-labelled cMBP radiotracers can be prepared and
subjected to sterile filtration without significant loss of
radiotracer due to adsorption to the filter.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates the FASTLAB.TM. chemistry synthesizer platform
configuration for Example 11.
DETAILED DESCRIPTION OF THE INVENTION
In a first aspect, the present invention provides an imaging agent
which comprises an .sup.18F-radiolabelled c-Met binding cyclic
peptide, wherein said c-Met binding cyclic peptide is an 18 to
30-mer cyclic peptide of Formula I: Z.sup.1-[cMBP]-Z.sup.2 (I)
where: cMBP is of Formula II: -(A).sub.x-Q-(A').sub.y- (II) where Q
is the amino acid sequence (SEQ ID NO: 1):
TABLE-US-00002
-Cys.sup.a-X.sup.1-Cys.sup.c-X.sup.2-Gly-Pro-Pro-X.sup.3-Phe-Glu-Cys.sup.d-
-Trp-Cys.sup.b-Tyr-X.sup.4-X.sup.5-X.sup.6-
wherein X.sup.1 is Asn, H is or Tyr; X.sup.2 is Gly, Ser, Thr or
Asn; X.sup.3 is Thr or Arg; X.sup.4 is Ala, Asp, Glu, Gly or Ser;
X.sup.5 is Ser or Thr; X.sup.6 is Asp or Glu; and Cys.sup.a-d are
each cysteine residues such that residues a and b as well as c and
d are cyclised to form two separate disulfide bonds; A and A' are
independently any amino acid other than Cys, with the proviso that
at least one of A and A' is present and is Lys; x and y are
independently integers of value 0 to 13, and are chosen such that
[x+y]=1 to 13; Z.sup.1 is attached to the N-terminus of cMBP, and
is H or M.sup.IG; Z.sup.2 is attached to the C-terminus of cMBP and
is OH, OB.sup.c, or M.sup.IG, where B.sup.c is a biocompatible
cation; each M.sup.IG is independently a metabolism inhibiting
group which is a biocompatible group which inhibits or suppresses
in vivo metabolism of the cMBP peptide; wherein cMBP is labelled at
the Lys residue of the A or A' groups with .sup.18F.
By the term "imaging agent" is meant a compound suitable for
imaging the mammalian body. Preferably, the mammal is an intact
mammalian body in vivo, and is more preferably a human subject.
Preferably, the imaging agent can be administered to the mammalian
body in a minimally invasive manner, i.e. without a substantial
health risk to the mammalian subject when carried out under
professional medical expertise. Such minimally invasive
administration is preferably intravenous administration into a
peripheral vein of said subject, without the need for local or
general anaesthetic.
The term "in vivo imaging" as used herein refers to those
techniques that non-invasively produce images of all or part of an
internal aspect of a mammalian subject.
By the term "c-Met binding cyclic peptide" is meant a peptide which
binds to the hepatocyte growth factor receptor, also known as c-Met
(or simply MET). Suitable such peptides of the present invention
are cyclic peptides of 18 to 30 amino acids of Formula I. Such
peptides have an apparent K.sub.D for c-Met of less than about 20
nM. The cMBP sequence of said peptides comprises proline residues,
and it is known that such residues can exhibit cis/trans
isomerisation of the backbone amide bond. The cMBP peptides of the
present invention include any such isomers.
The Z.sup.1 group substitutes the amine group of the last amino
acid residue of the cMBP, i.e., the amino terminus. Thus, when
Z.sup.1 is H, the amino terminus of the cMBP terminates in a free
NH.sub.2 group of the last amino acid residue. The Z.sup.2 group
substitutes the carbonyl group of the last amino acid residue of
the cMBP--i.e. the carboxy terminus. Thus, when Z.sup.2 is OH, the
carboxy terminus of the cMBP terminates in the free CO.sub.2H group
of the last amino acid residue, and when Z.sup.2 is OB.sup.c that
terminal carboxy group is ionised as a CO.sub.2B.sup.c group.
By the term "biocompatible cation" (B.sup.c) is meant a positively
charged counterion which forms a salt with an ionised, negatively
charged group, where said positively charged counterion is also
non-toxic and hence suitable for administration to the mammalian
body, especially the human body. Examples of suitable biocompatible
cations include: the alkali metals sodium or potassium; the
alkaline earth metals calcium and magnesium; and the ammonium ion.
Preferred biocompatible cations are sodium and potassium, most
preferably sodium.
By the term "metabolism inhibiting group" (M.sup.IG) is meant a
biocompatible group which inhibits or suppresses in vivo metabolism
of the cMBP peptide at either the amino terminus (Z.sup.1) or
carboxy terminus (Z.sup.2). Such groups are well known to those
skilled in the art and are suitably chosen from, for the peptide
amine terminus: N-acylated groups --NH(C.dbd.O)R.sup.G where the
acyl group --(C.dbd.O)R.sup.G has R.sup.G chosen from: C.sub.1-6
alkyl, or C.sub.3-10 aryl groups or comprises a polyethyleneglycol
(PEG) building block. For the peptide carboxy terminus:
carboxamide, tert-butyl ester, benzyl ester, cyclohexyl ester,
amino alcohol or a polyethyleneglycol (PEG) building block.
Preferred such PEG groups are the biomodifiers of Formula IA or
IB:
##STR00001##
17-amino-5-oxo-6-aza-3,9,12,15-tetraoxaheptadecanoic acid of
Formula IA
wherein p is an integer from 1 to 10. Alternatively, a PEG-like
structure based on a propionic acid derivative of Formula IB can be
used:
##STR00002## where p is as defined for Formula IA and q is an
integer from 3 to 15.
In Formula IB, p is preferably 1 or 2, and q is preferably 5 to
12.
Preferred such amino terminus M.sup.IG groups are acetyl,
benzyloxycarbonyl or trifluoroacetyl, most preferably acetyl.
By the term ".sup.18F-radiolabelled" is meant that the c-Met
binding cyclic peptide has covalently conjugated thereto the
radioisotope .sup.18F. The .sup.18F is suitably attached via a C--F
fluoroalkyl or fluoroaryl bond, since such bonds are relatively
stable in vivo, and hence confer resistance to metabolic cleavage
of the .sup.18F radiolabel from the cMBP peptide. The .sup.18F is
preferably attached via a C--F fluoroaryl bond. The .sup.18F may be
attached directly to one of the amino acids of the cMBP, but is
preferably conjugated as part of a radiofluorinated substituent on
the cMBP. Said substituents are preferably of formula:
-(L).sub.n-.sup.18F where: L is a synthetic linker group of formula
-(A1).sub.m- wherein each A1 is independently --CR.sub.2--,
--CR.dbd.CR--, --C.ident.C--, --CR.sub.2CO.sub.2--,
--CO.sub.2CR.sub.2--, --NR(C.dbd.O)--, --(C.dbd.O)NR--,
--NR(C.dbd.O)NR--, --NR(C.dbd.S)NR--, --SO.sub.2NR--,
--NRSO.sub.2--CR.sub.2OCR.sub.2--, --CR.sub.2SCR.sub.2--,
--CR.sub.2NRCR.sub.2--, --CR.sub.2--O--N.dbd., --CR.sub.2--O--NR--,
--CR.sub.2--O--NH(CO)--, a C.sub.4-8 cycloheteroalkylene group, a
C.sub.4-8 cycloalkylene group, a C.sub.5-12 arylene group, or a
C.sub.3-12 heteroarylene group, an amino acid, a sugar or a
monodisperse polyethyleneglycol (PEG) building block; each R is
independently chosen from H, C.sub.1-4 alkyl, C.sub.2-4 alkenyl,
C.sub.2-4 alkynyl, C.sub.1-4 alkoxyalkyl or C.sub.1-4 hydroxyalkyl;
m is an integer of value 1 to 20; n is an integer of value 0 or
1.
By the term "amino acid" is meant an L- or D-amino acid, amino acid
analogue (eg. naphthylalanine) or amino acid mimetic which may be
naturally occurring or of purely synthetic origin, and may be
optically pure, i.e. a single enantiomer and hence chiral, or a
mixture of enantiomers. Conventional 3-letter or single letter
abbreviations for amino acids are used herein. Preferably the amino
acids of the present invention are optically pure. By the term
"amino acid mimetic" is meant synthetic analogues of naturally
occurring amino acids which are isosteres, i.e. have been designed
to mimic the steric and electronic structure of the natural
compound. Such isosteres are well known to those skilled in the art
and include but are not limited to depsipeptides, retro-inverso
peptides, thioamides, cycloalkanes or 1,5-disubstituted tetrazoles
[see M. Goodman, Biopolymers, 24, 137, (1985)].
By the term "peptide" is meant a compound comprising two or more
amino acids, as defined above, linked by a peptide bond (i.e. an
amide bond linking the amine of one amino acid to the carboxyl of
another).
By the term "sugar" is meant a mono-, di- or tri-saccharide.
Suitable sugars include: glucose, galactose, maltose, mannose, and
lactose. Optionally, the sugar may be functionalised to permit
facile coupling to amino acids. Thus, eg. a glucosamine derivative
of an amino acid can be conjugated to other amino acids via peptide
bonds. The glucosamine derivative of asparagine (commercially
available from NovaBiochem) is one example of this:
##STR00003##
When A and A' are "any amino acid other than Cys" that means that
the additional amino acid of the A and A' groups lack free thiol
groups, in particular Cys residues. That is because an additional
Cys residue would risk disulfide bridge scrambing with the
Cys.sup.a-Cys.sup.b and Cys.sup.c-Cys.sup.d disulfide bridges of
the Q sequence, with consequent loss or reduction of c-Met binding
affinity.
Preferred Features.
Preferred cMBP peptides of the present invention have a K.sub.D for
binding of c-Met to c-Met/HGF complex of less than about 10 nM
(based on fluorescence polarisation assay measurements), most
preferably in the range 1 to 5 nM, with less than 3 nM being the
ideal.
The cMBP peptide of Formulae I and II is preferably of Formula IIA:
-(A).sub.x-Q-(A').sub.z-Lys- (IIA) wherein A is as defined for
Formula II, z is an integer of value 0 to 12, and [x+z]=0 to 12,
and cMBP comprises only one Lys residue.
Thus, in Formula IIA the single Lys residue is located specifically
at the C-terminus of the cMBP. That in turn means that the .sup.18F
radiolabel is preferably located at the C-terminus position.
Q preferably comprises the amino acid sequence of either SEQ ID NO:
2 or SEQ ID NO: 3:
TABLE-US-00003 (SEQ-2)
Ser-Cys.sup.a-X.sup.1-Cys.sup.c-X.sup.2-Gly-Pro-Pro-X.sup.3-Phe-Glu-Cys.su-
p.d-Trp-Cys.sup.b-Tyr-X.sup.4-X.sup.5-X.sup.6; (SEQ-3)
Ala-Gly-Ser-Cys.sup.a-X.sup.1-Cys.sup.c-X.sup.2-Gly-Pro-Pro-X.sup.3-Phe-Gl-
u-Cys.sup.d-Trp-Cys.sup.b-Tyr- X.sup.4-X.sup.5-X.sup.6-Gly-Thr.
In SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, X.sup.3 is
preferably Arg. In Formula I and Formula II, the -(A).sub.x- or
-(A').sub.y-groups preferably comprise a linker peptide which is
chosen from:
TABLE-US-00004 (SEQ ID NO: 4) -Gly-Gly-Gly-Lys-, (SEQ ID NO: 5)
-Gly-Ser-Gly-Lys- or (SEQ ID NO: 6) -Gly-Ser-Gly-Ser-Lys-.
The cMBP peptide of the first aspect preferably has the amino acid
sequence (SEQ ID NO: 7):
TABLE-US-00005
Ala-Gly-Ser-Cys.sup.a-Tyr-Cys.sup.c-Ser-Gly-Pro-Pro-Arg-Phe-Glu-Cys.sup.d--
Trp-Cys.sup.b-Tyr- Glu-Thr-Glu-Gly-Thr-Gly-Gly-Gly-Lys.
Preferred imaging agents of the present invention have both cMBP
peptide termini protected by M.sup.IG groups, i.e. preferably both
Z.sup.1 and Z.sup.2 are M.sup.IG, which will usually be different.
Having both peptide termini protected in this way is important for
in vivo imaging applications, since otherwise rapid peptide
metabolism would be expected with consequent loss of selective
binding affinity for c-Met. When both Z.sup.1 and Z.sup.2 are
M.sup.IG, preferably Z.sup.1 is acetyl and Z.sup.2 is a primary
amide. Most preferably, Z.sup.1 is acetyl and Z.sup.2 is a primary
amide and the .sup.18F moiety is attached to the epsilon amine side
chain of a lysine residue of cMBP.
The radiofluorinated substituent -(L).sub.n-.sup.18F may be
attached to the alpha amino group of the N-terminus of the c-Met
binding peptide, or alternatively to the amine side chain of any
amino-substituted amino acids (e.g. Lys residues). Preferably, it
is attached to the epsilon (.epsilon.) amine group of the Lys
residue of the cMBP.
Preferred radiofluorinated substituents -(L).sub.n-.sup.18F have
n=1, i.e. a synthetic linker group as defined above is present.
More preferred such substituents comprise the .sup.18F radiolabel
bound to a phenyl group, i.e. the substituent is of formula:
-(A1).sub.xC.sub.6H.sub.4-.sup.18F
where: A1 is as defined above, x is an integer of value 0 to 5.
Most preferred such substituents arise from either N-acylation of
the Lys amine residue with a fluorinated active ester, or
condensation of an amino-oxy derivative of the Lys amine residue
with a fluorinated benzaldehyde, and are of formula:
##STR00004##
The imaging agents of the first aspect can be prepared as described
in the fifth aspect (below).
In a second aspect, the present invention provides an imaging agent
composition which comprises: (i) the .sup.18F-radiolabelled c-Met
binding cyclic peptide of the first aspect; (ii) an unlabelled
c-Met binding cyclic peptide; wherein: said c-Met binding cyclic
peptide has the same amino acid sequence in (i) and (ii) and
wherein the unlabelled cMBP peptide is present in said composition
at no more than 50 times the molar amount of said .sup.18F-labelled
cMBP peptide.
Preferred embodiments of the .sup.18F-radiolabelled c-Met binding
cyclic peptide in the second aspect are as described in the first
aspect (above).
The term "composition" has its conventional meaning, i.e. a mixture
of the specified components. The composition may be in solid or
liquid/solution form.
By the term "unlabelled" is meant that the c-Met binding cyclic
peptide is non-radioactive, i.e. is not radiolabelled with
.sup.18F, or any other radioisotope. One or more such peptides may
be present in the composition, and such unlabelled peptides
primarily include the non-radioactive precursors of the fourth
aspect (below). The term `unlabelled` excludes the c-Met binding
cyclic peptide labelled with .sup.19F, where said .sup.19F is
present in the .sup.18F-fluoride used to radiolabel said c-Met
binding cyclic peptide and is thus a product of the same
radiolabelling reaction. As is known in the art, if two
fluorine-substituted compounds differ only in the isotopes of the
fluorine atom, they would behave chemically in an almost identical
manner, and hence their separation would be extremely difficult.
The unlabelled c-Met binding cyclic peptide or precursor preferably
has the groups Z.sup.1 and/or Z.sup.2 already attached. The present
inventors have found that, when an .sup.18F-labelled aldehyde is
used to conjugate to an aminooxy-functionalised cMBP peptide
precursor, that non-radioactive aldehydic impurities are the
principal sources of side-products. An important such aldehydic
impurity in .sup.18F-benzaldehyde is DMAP (i.e.
4-dimethylamino)benzaldehyde. Hence, the conjugation products of
non-radioactive aldehydes (such as DMAP) with
aminooxy-functionalised cMBP peptide are also within the scope of
the term `unlabelled c-Met binding cyclic peptide`.
Preferably, the unlabelled c-Met binding cyclic peptide is present
in said composition at up to 30, more preferably up to 20, most
preferably less than 10 times the molar amount of the corresponding
.sup.18F-labelled peptide.
The composition of the second aspect is preferably in solution
form, wherein the components (i) and (ii) are both present in
solution. More preferably, the solution is a biocompatible solvent,
or mixture of two or more such solvents. Preferred such
biocompatible solvents are described in the third aspect (below),
and preferably comprises an aqueous solvent.
The present inventors have found that, at radiotracer
concentrations--an approximate concentration range of 1 to 50
.mu.g/ml, the .sup.18F-labelled cMBP peptides of the invention
exhibit unwanted binding to a variety of materials. Since the
radiotracer is present at such very low concentration, even a small
chemical amount of adsorption can represent a significant
percentage of the radioisotope present. That radiotracer
concentration is to be compared with e.g. the corresponding cyanine
dye-labelled c-Met binding peptides, where the concentration would
be approximately 2 to 10 mg/ml--a factor of almost a thousand
higher. In such cases, loss of .mu.g amounts of material to
adsorption would be an insignificant percentage of the dye-labelled
peptides still in solution. The materials where radiotracer
adhesion have been observed include plastics, glass and silica. In
the case of filters, that can mean high percentage losses of
radioactivity when carrying out sterile filtration.
The present inventors have found that the above adhesion phenomenon
stems from the fact that the cMBP peptide precipitates under acidic
conditions (particularly at lower temperatures). It is therefore
preferred to maintain the composition at or above pH 7.5, more
preferably pH 8.0 or above in order to keep the desired
.sup.18F-labelled c-Met binding peptide in solution, and thus
avoiding loss of material. As an alternative to, or in addition to
the use of controlled pH, a solubiliser may be included.
By the term "solubiliser" is meant an additive present in the
composition which increases the solubility of the imaging agent in
the solvent. A preferred such solvent is aqueous media, and hence
the solubiliser preferably improves solubility in water. Suitable
such solubilisers include: C.sub.1-4 alcohols; glycerine;
polyethylene glycol (PEG); propylene glycol; polyoxyethylene
sorbitan monooleate; sorbitan monooloeate; polysorbates;
poly(oxyethylene)poly(oxypropylene)poly(oxyethylene) block
copolymers (PLURONICS.TM. brand poloxamers); cyclodextrins (e.g.
alpha, beta or gamma cyclodextrin,
hydroxypropyl-.beta.-cyclodextrin or
hydroxypropyl-.gamma.-cyclodextrin) and lecithin.
Preferred solubilisers are cyclodextrins, C.sub.1-4 alcohols and
PLURONICS.TM. brand poloxamers, more preferably cyclodextrins and
C.sub.2-4 alcohols. When the solubiliser is an alcohol, it is
preferably ethanol or propanol, more preferably ethanol. Ethanol
has a potential dual role, since it can also function as a
radioprotectant. When the solubiliser is a cyclodextrin, it is
preferably a cyclodextrin, more preferably
hydroxypropyl-.beta.-cyclodextrin (HPCD). The concentration of
cyclodextrin can be from about 0.1 to about 40 mg/mL, preferably
between about 5 and about 35 mg/mL, more preferably 20 to 30 mg/ml,
most preferably around 25 mg/ml. When a single solubiliser is used,
it is preferably ethanol or hydroxypropyl-.beta.-cyclodextrin, more
preferably ethanol. When a combination of solubilisers is used, it
is preferably ethanol and to hydroxypropyl-.beta.-cyclodextrin.
Preferably, the composition of the second aspect is maintained at
pH at or above 7.5, optionally with 5-10% v/v ethanol as
solubiliser.
The imaging agent composition of the second aspect preferably
further comprises one or more radioprotectants. By the term
"radioprotectant" is meant a compound which inhibits degradation
reactions, such as redox processes, by trapping highly-reactive
free radicals, such as oxygen-containing free radicals arising from
the radiolysis of water. A combination of two or more different
radioprotectants may be used. The radioprotectants of the present
invention are suitably chosen from: ethanol; ascorbic acid;
para-aminobenzoic acid (i.e. 4-aminobenzoic acid or pABA); gentisic
acid (i.e. 2,5-dihydroxybenzoic acid), and where applicable salts
of such acids with a biocompatible cation as define above. The
radioprotectant of the present invention preferably comprises
para-aminobenzoic acid or sodium para-aminobenzoate.
A most preferred imaging agent composition of the present invention
comprises the cMBP peptide of SEQ-7 having Z.sup.1=Z.sup.2=M.sup.IG
attached, and a combination of para-aminobenzoic acid
radioprotectant and ethanol radioprotectant/solubiliser in aqueous
buffer. A preferred peptide of SEQ-7 in such preferred compositions
is Peptide 1, and a preferred .sup.18F-labelled cMBP peptide is
Compound 3. The radioactive concentration is preferably less than
350 MBq/ml, with a pABA concentration of 2 mg/ml, and ethanol at
about 5-10% vol/vol, preferably 6.5-7.5% vol/vol.
In a third aspect, the present invention provides a pharmaceutical
composition which comprises the imaging agent of the first aspect,
or the imaging agent composition of the second aspect, together
with a biocompatible carrier, in a sterile form suitable for
mammalian administration.
Preferred aspects of the imaging agent and composition in the third
aspect are as defined in the first and second aspects
respectively.
The "biocompatible carrier" is a fluid, especially a liquid, in
which the imaging agent can be suspended or preferably dissolved,
such that the composition is physiologically tolerable, i.e. can be
administered to the mammalian body without toxicity or undue
discomfort. The biocompatible carrier is suitably an injectable
carrier liquid such as sterile, pyrogen-free water for injection;
an aqueous solution such as saline (which may advantageously be
balanced so that the final product for injection is isotonic); an
aqueous buffer solution comprising a biocompatible buffering agent
(e.g. phosphate buffer); an aqueous solution of one or more
tonicity-adjusting substances (eg. salts of plasma cations with
biocompatible counterions), sugars (e.g. glucose or sucrose), sugar
alcohols (eg. sorbitol or mannitol), glycols (eg. glycerol), or
other non-ionic polyol materials (eg. polyethyleneglycols,
propylene glycols and the like). Preferably the biocompatible
carrier is pyrogen-free water for injection, isotonic saline or
phosphate buffer. Use of a buffer is preferred in order to control
pH.
The imaging agents and biocompatible carrier are each supplied in
suitable vials or vessels which comprise a sealed container which
permits maintenance of sterile integrity and/or radioactive safety,
plus optionally an inert headspace gas (eg. nitrogen or argon),
whilst permitting addition and withdrawal of solutions by syringe
or cannula. A preferred such container is a septum-sealed vial,
wherein the gas-tight closure is crimped on with an overseal
(typically of aluminium). The closure is suitable for single or
multiple puncturing with a hypodermic needle (e.g. a crimped-on
septum seal closure) whilst maintaining sterile integrity. Such
containers have the additional advantage that the closure can
withstand vacuum if desired (eg. to change the headspace gas or
degas solutions), and withstand pressure changes such as reductions
in pressure without permitting ingress of external atmospheric
gases, such as oxygen or water vapour.
Preferred multiple dose containers comprise a single bulk vial
(e.g. of 10 to 30 cm.sup.3 volume) which contains multiple patient
doses, whereby single patient doses can thus be withdrawn into
clinical grade syringes at various time intervals during the viable
lifetime of the preparation to suit the clinical situation.
Pre-filled syringes are designed to contain a single human dose, or
"unit dose" and are therefore preferably a disposable or other
syringe suitable for clinical use. The pharmaceutical compositions
of the present invention preferably have a dosage suitable for a
single patient and are provided in a suitable syringe or container,
as described above.
The pharmaceutical composition may contain additional optional
excipients such as: an antimicrobial preservative, pH-adjusting
agent, filler, radioprotectant, solubiliser or osmolality adjusting
agent. The terms "radioprotectant" and "solubiliser" and preferred
embodiments thereof are as described in the second aspect (above).
By the term "antimicrobial preservative" is meant an agent which
inhibits the growth of potentially harmful micro-organisms such as
bacteria, yeasts or moulds. The antimicrobial preservative may also
exhibit some bactericidal properties, depending on the dosage
employed. The main role of the antimicrobial preservative(s) of the
present invention is to inhibit the growth of any such
micro-organism in the pharmaceutical composition. The antimicrobial
preservative may, however, also optionally be used to inhibit the
growth of potentially harmful micro-organisms in one or more
components of kits used to prepare said composition prior to
administration. Suitable antimicrobial preservative(s) include: the
parabens, i.e. methyl, ethyl, propyl or butyl paraben or mixtures
thereof; benzyl alcohol; phenol; cresol; cetrimide and thiomersal.
Preferred antimicrobial preservative(s) are the parabens.
The term "pH-adjusting agent" means a compound or mixture of
compounds useful to ensure that the pH of the composition is within
acceptable limits (approximately pH 4.0 to 10.5) for human or
mammalian administration. Suitable such pH-adjusting agents include
pharmaceutically acceptable buffers, such as tricine, phosphate or
TRIS [i.e. tris(hydroxymethyl)aminomethane], and pharmaceutically
acceptable bases such as sodium carbonate, sodium bicarbonate or
mixtures thereof. When the composition is employed in kit form, the
pH adjusting agent may optionally be provided in a separate vial or
container, so that the user of the kit can adjust the pH as part of
a multi-step procedure.
By the term "filler" is meant a pharmaceutically acceptable bulking
agent which may facilitate material handling during production and
lyophilisation. Suitable fillers include inorganic salts such as
sodium chloride, and water soluble sugars or sugar alcohols such as
sucrose, maltose, mannitol or trehalose.
The pharmaceutical compositions of the third aspect may be prepared
under aseptic manufacture (i.e. clean room) conditions to give the
desired sterile, non-pyrogenic product. It is preferred that the
key components, especially the associated reagents plus those parts
of the apparatus which come into contact with the imaging agent
(eg. vials) are sterile. The components and reagents can be
sterilised by methods known in the art, including: sterile
filtration, terminal sterilisation using e.g. gamma-irradiation,
autoclaving, dry heat or chemical treatment (e.g. with ethylene
oxide). It is preferred to sterilise some components in advance, so
that the minimum number of manipulations needs to be carried out.
As a precaution, however, it is preferred to include at least a
sterile filtration step as the final step in the preparation of the
pharmaceutical composition.
As noted above, the pharmaceutical compositions of the present
invention are preferably maintained at pH 7.5 or above and/or
comprise a solubiliser, so that a sterile filtration step may be
used without undue loss of radioactivity adsorbed to the filter
material. Similar considerations apply to manipulations of the
pharmaceutical compositions in clinical grade syringes, or using
plastic tubing, where adsorption may cause loss of radioactivity
without the use of a solubiliser.
The pharmaceutical composition is preferably prepared as described
in the sixth aspect (below).
The pharmaceutical composition of the third aspect may optionally
be prepared from a kit. Such kits comprise the c-Met binding
peptide of Formula I as described in the first aspect or the
precursor of the fourth aspect in sterile, apyrogenic form such
that, upon reaction with a sterile supply of the radioisotope
.sup.18F in a suitable solvent, radiolabelling occurs to give the
desired .sup.18F-labelled c-Met binding peptide.
For the kit, the c-Met binding peptide or precursor, plus other
optional excipients as described above, are preferably provided as
a lyophilised powder in a suitable vial or container. The agent is
then designed to be reconstituted either with .sup.18F in a
biocompatible carrier directly (i.e. as a reconstitution), or first
reconstitution of the kit with a biocompatible carrier, followed by
reaction with a supply of .sup.18F.
A preferred sterile form of the c-Met binding peptide or precursor
is a lyophilised solid. The sterile, solid form is preferably
supplied in a pharmaceutical grade container, as described for the
pharmaceutical composition (above). When the kit is lyophilised,
the formulation may optionally comprise a cryoprotectant chosen
from a saccharide, preferably mannitol, maltose or tricine.
In a fourth aspect, the present invention provides a precursor,
useful in the preparation of the .sup.18F-radiolabelled c-Met
binding cyclic peptide of the third aspect, or the composition of
the first or second aspects, which comprises: (i) the c-Met binding
cyclic peptide of Formula I as defined in the first aspect, wherein
Z.sup.1=Z.sup.2=M.sup.IG; or (ii) an amino-oxy functionalised c-Met
binding cyclic peptide.
By the term "amino-oxy functionalised c-Met binding cyclic peptide"
is meant the c-Met binding cyclic peptide of Formula I having
covalently conjugated thereto an amino-oxy functional group. Such
amino-oxy groups are of formula --O--NH.sub.2, preferably
--CH.sub.2O--NH.sub.2 and have the advantage that the amine of the
amino-oxy group is more reactive than a Lys amine group in
condensation reactions with aldehydes to form oxime ethers. Such
amino-oxy groups are suitably attached at the Lys residue of the
cMBP, as described below.
The precursor is non-radioactive, and is designed so that it can be
obtained in a high degree of chemical purity. It is also designed
so that, upon reaction with a suitable source of .sup.18F, reaction
occurs efficiently with satisfactory radiochemical purity (RCP).
The "suitable source of .sup.18F" depends on the nature of the
precursor. When the precursor comprises the unlabelled c-Met
binding peptide of Formula I, the amine group of the lysine (Lys)
residue of the unlabelled peptide is designed to be the site of
radiolabelling. The termini of the cMBP peptide are protected,
since Z.sup.1=Z.sup.2=M.sup.IG. Preferred such c-Met binding
peptides and preferred Z.sup.1/Z.sup.2 groups are as described in
the first aspect. Thus, the suitable source of .sup.18F is designed
to react as efficiently as possible with the lysine amine group,
preferably the Lys epsilon amine.
For the preparation of the pharmaceutical composition of the third
aspect, the precursor is preferably in sterile form, more
preferably a lyophilised solid.
The precursor of the fourth aspect is preferably an amino-oxy
functionalised c-Met binding peptide.
c-Met binding peptides of Formula I, i.e. Z.sup.1-[cMBP]-Z.sup.2 of
the present invention may be obtained by a method of preparation
which comprises: (i) solid phase peptide synthesis of a linear
peptide which has the same peptide sequence as the desired cMBP
peptide and in which the Cys.sup.a and Cys.sup.b are unprotected,
and the Cys.sup.c and Cys.sup.d residues have thiol-protecting
groups; (ii) treatment of the peptide from step (i) with aqueous
base in solution to give a monocyclic peptide with a first
disulphide bond linking Cys.sup.a and Cys.sup.b; (iii) removal of
the Cys.sup.c and Cys.sup.d thiol-protecting groups and cyclisation
to give a second disulphide bond linking Cys.sup.c and Cys.sup.d,
which is the desired bicyclic peptide product
Z.sup.1-[cMBP]-Z.sup.2.
By the term "protecting group" is meant a group which inhibits or
suppresses undesirable chemical reactions, but which is designed to
be sufficiently reactive that it may be cleaved from the functional
group in question under mild enough conditions that do not modify
the rest of the molecule. After deprotection the desired product is
obtained. Amine protecting groups are well known to those skilled
in the art and are suitably chosen from: Boc (where Boc is
tert-butyloxycarbonyl), Fmoc (where Fmoc is
fluorenylmethoxycarbonyl), trifluoroacetyl, allyloxycarbonyl, Dde
[i.e. 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl] or Npys (i.e.
3-nitro-2-pyridine sulfenyl). Suitable thiol protecting groups are
Trt (Trityl), Acm (acetamidomethyl), t-Bu (tert-butyl),
tert-Butylthio, methoxybenzyl, methylbenzyl or Npys
(3-nitro-2-pyridine sulfenyl). The use of further protecting groups
are described in `Protective Groups in Organic Synthesis`, 4.sup.th
Edition, Theorodora W. Greene and Peter G. M. Wuts, [Wiley
Blackwell, (2006)]. Preferred amine protecting groups are Boc and
Fmoc, most preferably Boc. Preferred amine protecting groups are
Trt and Acm.
Examples 1 and 2 provide further specific details. Further details
of solid phase peptide synthesis are described in P.
Lloyd-Williams, F. Albericio and E. Girald; Chemical Approaches to
the Synthesis of Peptides and Proteins, CRC Press, 1997. The cMBP
peptides are best stored under inert atmosphere and kept in a
freezer. When used in solution, it is best to avoid pH above 7
since that risks scrambling of the disulfide bridges.
Amino-oxy functionalised c-Met binding peptides can be prepared by
the methods of Poethko et al [J. Nucl. Med., 45, 892-902 (2004)],
Schirrmacher et al [Bioconj. Chem., 18, 2085-2089 (2007)],
Solbakken et al [Bioorg. Med. Chem. Lett, 16, 6190-6193 (2006)] or
Glaser et al [Bioconj. Chem., 19, 951-957 (2008)]. The amino-oxy
group may optionally be conjugated in two steps. First, the
N-protected amino-oxy carboxylic acid or N-protected amino-oxy
activated ester is conjugated to the c-Met binding peptide. Second,
the intermediate N-protected amino-oxy functionalised c-Met binding
peptide is deprotected to give the desired product [see Solbakken
and Glaser papers cited above]. N-protected amino-oxy carboxylic
acids such as Boc-NH--O--CH.sub.2(C.dbd.O)OH are commercially
available, e.g. from Novabiochem.
In a fifth aspect, the present invention provides a method of
preparation of the .sup.18F-radiolabelled c-Met binding cyclic
peptide of the first aspect, which comprises: (i) provision of the
precursor of the fourth aspect; (ii) when said precursor comprises
an unlabelled c-Met binding cyclic peptide of Formula I wherein
Z.sup.1=Z.sup.2 reaction with either an .sup.18F-labelled activated
ester, or an .sup.18F-labelled carboxylic acid in the presence of
an activating agent, to give the .sup.18F-radiolabelled c-Met
binding cyclic peptide conjugated via an amide linkage at the Lys
residue of the cMBP of said cyclic peptide; (iii) when said
precursor comprises an amino-oxy functionalised c-Met binding
cyclic peptide, reaction with either: (a) an .sup.18F-labelled
activated ester, or an .sup.18F-labelled carboxylic acid in the
presence of an activating agent, to give the .sup.18F-radiolabelled
c-Met binding cyclic peptide conjugated via an amide linkage at the
amino-oxy position of said functionalised peptide; or (b) an
.sup.18F-labelled aldehyde to give the .sup.18F-radiolabelled c-Met
binding cyclic peptide conjugated via an oxime ether linkage at the
amino-oxy position of said functionalised peptide.
By the term "activated ester" or "active ester" is meant an ester
derivative of the associated carboxylic acid which is designed to
be a better leaving group, and hence permit more facile reaction
with nucleophile, such as amines. Examples of suitable active
esters are: N-hydroxysuccinimide (NETS); sulfo-succinimidyl ester;
pentafluorophenol; pentafluorothiophenol; para-nitrophenol;
hydroxybenzotriazole and PyBOP (i.e.
benzotriazol-1-yl-oxytripyrrolidinophosphonium
hexafluorophosphate). Preferred active esters are
N-hydroxysuccinimide or pentafluorophenol esters, especially
N-hydroxysuccinimide esters.
By the term "activating agent" is meant a reagent used to
facilitate coupling between an amine and a carboxylic acid to
generate an amide. Suitable such activating agents are known in the
art and include carbodiimides such as EDC
[N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide and
N,N'-dialkylcarbodiimides such as dicyclohexylcarbodiimide or
diisopropylcarbodiimide; and triazoles such as HBTU
[O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate], HATU
[O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate], and PyBOP
[benzotriazol-1-yloxy)tripyrrolidinophosphonium
hexafluorophosphate]. Further details are given in "March's
Advanced Organic Chemistry", 5.sup.th Edition, pages 508-510, Wiley
Interscience (2001). A preferred such activating agent is EDC.
.sup.18F-labelled activated esters, such as [.sup.18F]SFB can be
prepared by the method of Glaser et al, and references therein [J.
Lab. Comp. Radiopharm., 52, 327-330 (2009)], or the automated
method of Marik et al [Appl. Rad. Isot., 65(2), 199-203
(2007)]:
##STR00005##
.sup.18F-labelled carboxylic acids can be obtained by the method of
Marik et al cited above. .sup.18F-labelled aliphatic aldehydes of
formula
.sup.18F(CH.sub.2).sub.2O[CH.sub.2CH.sub.2O].sub.qCH.sub.2CHO,
where q is 3, can be obtained by the method of Glaser et al
[Bioconj. Chem., 19(4), 951-957 (2008)].
.sup.18F-fluorobenzaldehyde can be obtained by the method of Glaser
et al [J. Lab. Comp. Radiopharm., 52, 327-330 (2009)]. The
precursor Me.sub.3N.sup.+--C.sub.6H.sub.4--CHO.
CF.sub.3SO.sub.3.sup.- is obtained by the method of Haka et al [J.
Lab. Comp. Radiopharm., 27, 823-833 (1989)].
The conjugation of .sup.18F-labelled aldehydes to amino-oxy
functionalised c-Met peptides is preferably carried out in the
presence of an aniline catalyst as described by Flavell et al [J.
Am. Chem. Soc., 130(28), 9106-9112 (2008)]. Whilst it is possible
to use protected amino-oxy c-Met peptides (such as Compound 1) as
precursors, the free amino-oxy derivative (such as Compound 2) is
preferred. That is because the whole synthesis is more amenable to
automation, whereas with the protected precursor, a manual
deprotection step is typically required.
In a sixth aspect, the present invention provides a method of
preparation of the imaging agent composition of the second aspect,
or the pharmaceutical composition thereof of the third aspect,
which comprises: (i) preparing the .sup.18F-radiolabelled c-Met
binding cyclic peptide as defined in the method of preparation of
the fifth aspect; (ii) chromatographic separation of the unlabelled
c-Met binding cyclic peptide from the .sup.18F-radiolabelled c-Met
binding cyclic peptide.
Preferred aspects of the imaging agent composition and
pharmaceutical composition in the sixth aspect are as described in
the second and third aspect respectively.
The chromatographic separation of step (ii) may be carried out by
HPLC or SPE (solid phase extraction) using one or more SPE
cartridge(s). SPE is preferred when an automated synthesizer is
used, and HPLC is preferred in other circumstances. Example 5
provides a suitable HPLC method for Compound 3 of the present
invention.
The method of the sixth aspect is preferably used to obtain the
pharmaceutical composition of the third aspect. When the method is
used to provide the pharmaceutical composition of the third aspect,
the method of preparation is preferably carried out using an
automated synthesizer apparatus.
By the term "automated synthesizer" is meant an automated module
based on the principle of unit operations as described by
Satyamurthy et al [Clin. Positr. Imag., 2(5), 233-253 (1999)]. The
term `unit operations` means that complex processes are reduced to
a series of simple operations or reactions, which can be applied to
a range of materials. Such automated synthesizers are preferred for
the method of the present invention especially when a
radiopharmaceutical composition is desired. They are commercially
available from a range of suppliers [Satyamurthy et al, above],
including: GE Healthcare; CTI Inc; Ion Beam Applications S.A.
(Chemin du Cyclotron 3, B-1348 Louvain-La-Neuve, Belgium); Raytest
(Germany) and Bioscan (USA).
Commercial automated synthesizers also provide suitable containers
for the liquid radioactive waste generated as a result of the
radiopharmaceutical preparation. Automated synthesizers are not
typically provided with radiation shielding, since they are
designed to be employed in a suitably configured radioactive work
cell. The radioactive work cell provides suitable radiation
shielding to protect the operator from potential radiation dose, as
well as ventilation to remove chemical and/or radioactive vapours.
The automated synthesizer preferably comprises a cassette. By the
term "cassette" is meant a piece of apparatus designed to fit
removably and interchangeably onto an automated synthesizer
apparatus (as defined below), in such a way that mechanical
movement of moving parts of the synthesizer controls the operation
of the cassette from outside the cassette, i.e. externally.
Suitable cassettes comprise a linear array of valves, each linked
to a port where reagents or vials can be attached, by either needle
puncture of an inverted septum-sealed vial, or by gas-tight,
marrying joints. Each valve has a male-female joint which
interfaces with a corresponding moving arm of the automated
synthesizer. External rotation of the arm thus controls the opening
or closing of the valve when the cassette is attached to the
automated synthesizer. Additional moving parts of the automated
synthesizer are designed to clip onto syringe plunger tips, and
thus raise or depress syringe barrels.
The cassette is versatile, typically having several positions where
reagents can be attached, and several suitable for attachment of
syringe vials of reagents or chromatography cartridges (eg. SPE).
The cassette always comprises a reaction vessel. Such reaction
vessels are preferably 1 to 10 cm.sup.3, most preferably 2 to 5
cm.sup.3 in volume and are configured such that 3 or more ports of
the cassette are connected thereto, to permit transfer of reagents
or solvents from various ports on the cassette. Preferably the
cassette has 15 to 40 valves in a linear array, most preferably 20
to 30, with 25 being especially preferred. The valves of the
cassette are preferably each identical, and most preferably are
3-way valves. The cassettes are designed to be suitable for
radiopharmaceutical manufacture and are therefore manufactured from
materials which are of pharmaceutical grade and ideally also are
resistant to radiolysis.
Preferred automated synthesizers of the present invention are those
which comprise a disposable or single use cassette which comprises
all the reagents, reaction vessels and apparatus necessary to carry
out the preparation of a given batch of radiofluorinated
radiopharmaceutical. The cassette means that the automated
synthesizer has the flexibility to be capable of making a variety
of different radiopharmaceuticals with minimal risk of
cross-contamination, by simply changing the cassette. The cassette
approach also has the advantages of: simplified set-up hence
reduced risk of operator error; improved GMP (Good Manufacturing
Practice) compliance; multi-tracer capability; rapid change between
production runs; pre-run automated diagnostic checking of the
cassette and reagents; automated barcode cross-check of chemical
reagents vs the synthesis to be carried out; reagent traceability;
single-use and hence no risk of cross-contamination, tamper and
abuse resistance.
Included in this aspect of the invention, is the use of an
automated synthesizer apparatus to prepare the pharmaceutical
composition of the second aspect.
In a seventh aspect, the present invention provides a method of
imaging of the mammalian body in vivo to obtain images of sites of
c-Met over-expression or localisation, which method comprises
imaging said body to which the imaging agent of the first aspect,
the imaging agent composition of the second aspect or the
pharmaceutical composition of the third aspect, had been previously
administered.
Preferred aspects of the imagine agent, imaging agent composition
and pharmaceutical composition in the seventh aspect are as
described in the first, second and third aspects respectively.
Preferably, the mammal is an intact mammalian body in vivo, and is
more preferably a human subject. Preferably, the imaging agent can
be administered to the mammalian body in a minimally invasive
manner, i.e. without a substantial health risk to the mammalian
subject even when carried out under professional medical expertise.
Such minimally invasive administration is preferably intravenous
administration into a peripheral vein of said subject, without the
need for local or general anaesthetic.
Preferably, the pharmaceutical composition of the third aspect is
used. In the method of imaging of the seventh aspect, the site of
c-Met over-expression or localisation is preferably a cancerous
tumour or metastasis. It is envisaged that the agent would be
useful for imaging c-Met expression in both precancerous and
cancerous tumours or metastases, potentially enabling selection of
therapy. In addition, repeated imaging with such an imaging agent
also has the potential to quickly and effectively monitor an
individual subject's response to established and novel therapeutic
regimens, thereby allowing discontinuation of ineffective
treatment. Furthermore, as overexpression of c-Met is a potential
attribute of aggressive tumours, it is anticipated that the c-Met
imaging agent has the potential to discriminate between aggressive
and less aggressive cancers at an early stage in their
development.
Included in this aspect is a method of diagnosis of sites of c-Met
over-expression or localisation within the mammalian body in vivo,
which comprises the imaging method of the seventh aspect.
The invention is illustrated by the non-limiting Examples detailed
below. Example 1 provides the synthesis of a cMBP peptide of the
invention having metabolism inhibiting groups
(Z.sup.1=Z.sup.2=M.sup.IG) at both termini (Peptide 1). Example 2
provides the synthesis of a protected precursor of the invention
(Compound 1). Example 3 provides the synthesis of the
non-radioactive, fluorinated (i.e. .sup.19F) counterpart of the
fluorine-labelled c-Met peptide (Compound 3A). Example 4 provides
the synthesis of an .sup.18F-radiofluorinated c-Met peptide of the
invention (Compound 3B). Example 5 provides HPLC conditions for the
separation of labelled and unlabelled c-Met binding peptides.
Example 6 provides the biodistribution of an .sup.18F-labelled
peptide of the invention (Compound 3B) in an animal tumour model.
The results show binding to the human c-Met receptor expressed in
the HT-29 tumours, and hence utility for tumour imaging. Example 7
demonstrates that the tumour uptake of Example 6 is specific, since
the uptake can be inhibited by co-administration of non-radioactive
.sup.19F-labelled c-Met binding peptide (Compound 3A). Example 8
also demonstrates reduced liver uptake of about 40% in primates
when .sup.19F-labelled c-Met binding peptide is co-administered.
Co-administration of a .sup.19F-labelled scrambled version of the
peptide, which has no affinity for the c-Met receptor, did not
significantly reduce the liver uptake. The liver has a high level
of c-Met expression, and the reduction in uptake following
competition with .sup.19F-labelled cMBP is therefore believed to
represent evidence of specific c-Met binding in vivo.
Example 9 shows that the solubiliser cyclodextrin has no
significant effect on the biodistribution of Compound 3B in vivo.
Example 10 compares the fully automated synthesis of Compound 3B
(starting from Compound 2), with the partially automated synthesis
starting from Compound 1. The use of Compound 2 is preferred
because it gives higher yields, and deprotection of Compound 1 has
the disadvantages of: (i) difficulty of establishing the degree of
deprotection prior to radiolabelling; (ii) the TFA used to
deprotect the peptide is not compatible with the plastic of the
automated synthesizer apparatus.
Example 11 provides the automated synthesis of Compound 3B, further
including automated use of SPE cartridge purification. The results
show that Compound 3B can be obtained in high purity and
satisfactory radiochemical yield using this approach. Example 12
provides the freeze-drying of a precursor of the invention. Example
13 demonstrates the effect of pH on an imaging agent of the
invention. Example 14 describes human imaging using an imaging
agent of the invention.
ABBREVIATIONS
Conventional single letter or 3-letter amino acid abbreviations are
used.
% id: percentage injected dose
Ac: Acetyl
Acm: Acetamidomethyl
ACN: Acetonitrile
Boc: tert-Butyloxycarbonyl
DCM: Dichloromethane
DIPEA: N,N-Diisopropylethyl amine
DMF: Dimethylformamide
DMSO: Dimethylsulfoxide
EDC: N-3-dimethylaminopropyl)-N'-ethylcarbodiimide.
Fmoc: 9-Fluorenylmethoxycarbonyl
HBTU: O-Benzotriazol-1-yl-N,N,N',N'-tetramethyluronium
hexafluorophosphate
HPLC: High performance liquid chromatography
HSPyU O--(N-succinimidyl)-N,N,N',N'-tetramethyleneuronium
hexafluorophosphate
NETS: N-hydroxy-succinimide
NMM: N-Methylmorpholine
NMP: 1-Methyl-2-pyrrolidinone
pABA: para-aminobenzoic acid.
Pbf: 2,2,4,6,7-Pentamethyldihydrobenzofuran-5-sulfonyl
PBS: Phosphate-buffered saline
p.i.: post-injection
PyBOP: benzotriazol-1-yl-oxytripyrrolidinophosphonium
hexafluorophosphate
tBu: tert-butyl
TFA: Trifluoroacetic acid
TIS: Triisopropylsilane
Trt: Trityl.
COMPOUNDS OF THE INVENTION
TABLE-US-00006 Name Structure Peptide 1 Disulfide bridges at
Cys4-16 and Cys6-14;
Ac-Ala-Gly-Ser-Cys-Tyr-Cys-Ser-Gly-Pro-Pro-Arg-
Phe-Glu-Cys-Trp-Cys-Tyr-Glu-Thr-Glu-Gly-Thr-
Gly-Gly-Gly-Lys-NH.sub.2 or Ac-AGSCYCSGPPRFECWCYETEGTGGGK-NH.sub.2
Compound 1 ##STR00006## Compound 2 ##STR00007## Compound 3
##STR00008## where: Compounds 1, 2 and 3 are functionalised at the
epsilon amine group of the carboxy terminal Lys of Peptide 1; Boc =
tert-Butyloxycarbonyl.
Example 1
Synthesis of Peptide 1
Step (a): Synthesis of Protected Precursor Linear Peptide.
The precursor linear peptide has the structure Ac-SEQ ID NO:
7-NH.sub.2:
Ac-Ala-Gly-Ser-Cys-Tyr-Cys(Acm)-Ser-Gly-Pro-Pro-Arg-Phe-Glu-Cys(Acm)-Trp-
-Cys-Tyr-Glu-Thr-Glu-Gly-Thr-Gly-Gly-Gly-Lys-NH.sub.2
The peptidyl resin
H-Ala-Gly-Ser(tBu)-Cys(Trt)-Tyr(tBu)-Cys(Acm)-Ser(tBu)-Gly-Pro-Pro-Arg(Pb-
f)-Phe-Glu(OtBu)-Cys(Acm)-Trp(Boc)-Cys(Trt)-Tyr(tBu)-Gly-Glu(OtBu)-Thr(.ps-
i..sup.Me,Mepro)-Glu(OtBu)-Gly-Thr(tBu)-Gly-Gly-Gly-Lys(Boc)-Polymer
was assembled on an Applied Biosystems 433A peptide synthesizer
using Fmoc chemistry starting with 0.1 mmol Rink Amide Novagel
resin. An excess of 1 mmol pre-activated amino acids (using HBTU)
was applied in the coupling steps. Glu-Thr pseudoproline
(Novabiochem 05-20-1122) was incorporated in the sequence. The
resin was transferred to a nitrogen bubbler apparatus and treated
with a solution of acetic anhydride (1 mmol) and NMM (1 mmol)
dissolved in DCM (5 mL) for 60 min. The anhydride solution was
removed by filtration and the resin washed with DCM and dried under
a stream of nitrogen.
The simultaneous removal of the side-chain protecting groups and
cleavage of the peptide from the resin was carried out in TFA (10
mL) containing 2.5% TIS, 2.5% 4-thiocresol and 2.5% water for 2
hours and 30 min. The resin was removed by filtration, TFA removed
in vacuo and diethyl ether added to the residue. The formed
precipitate was washed with diethyl ether and air-dried affording
264 mg of crude peptide.
Purification by preparative HPLC (gradient: 20-30% B over 40 min
where A=H.sub.2O/0.1% TFA and B=ACN/0.1% TFA, flow rate: 10 mL/min,
column: Phenomenex Luna 5.mu. C18 (2) 250.times.21.20 mm,
detection: UV 214 nm, product retention time: 30 min) of the crude
peptide afforded 100 mg of pure Peptide 1 linear precursor. The
pure product was analysed by analytical HPLC (gradient: 10-40% B
over 10 min where A=H.sub.2O/0.1% TFA and B=ACN/0.1% TFA, flow
rate: 0.3 mL/min, column: Phenomenex Luna 3.mu. C18 (2) 50.times.2
mm, detection: UV 214 nm, product retention time: 6.54 min).
Further product characterisation was carried out using electrospray
mass spectrometry (MH.sub.2.sup.2+ calculated: 1464.6,
MH.sub.2.sup.2+ found: 1465.1).
Step (b): Formation of Monocyclic Cys4-16 Disulfide Bridge.
Cys4-16;
Ac-Ala-Gly-Ser-Cys-Tyr-Cys(Acm)-Ser-Gly-Pro-Pro-Arg-Phe-Glu-Cys(-
Acm)-Trp-Cys-Tyr-Glu-Thr-Glu-Gly-Thr-Gly-Gly-Gly-Lys-NH.sub.2Ac-SEQ
ID NO: 7-NH.sub.2.
The linear precursor from step (a) (100 mg) was dissolved in 5%
DMSO/water (200 mL) and the solution adjusted to pH 6 using
ammonia. The reaction mixture was stirred for 5 days. The solution
was then adjusted to pH 2 using TFA and most of the solvent removed
by evaporation in vacuo. The residue (40 mL) was injected in
portions onto a preparative HPLC column for product
purification.
Purification by preparative HPLC (gradient: 0% B for 10 min, then
0-40% B over 40 min where A=H.sub.2O/0.1% TFA and B=ACN/0.1% TFA,
flow rate: 10 mL/min, column: Phenomenex Luna 5.mu. C18 (2)
250.times.21.20 mm, detection: UV 214 nm, product retention time:
44 min) of the residue afforded 72 mg of pure Compound 1 monocyclic
precursor.
The pure product (as a mixture of isomers P1 to P3) was analysed by
analytical HPLC (gradient: 10-40% B over 10 min where
A=H.sub.2O/0.1% TFA and B=ACN/0.1% TFA, flow rate: 0.3 mL/min,
column: Phenomenex Luna 3.mu. C18 (2) 50.times.2 mm, detection: UV
214 nm, product retention time: 5.37 min (P1); 5.61 min (P2); 6.05
min (P3)). Further product characterisation was carried out using
electrospray mass spectrometry (MH.sub.2.sup.2+ calculated: 1463.6,
MH.sub.2.sup.2+ found: 1464.1 (P1); 1464.4 (P2); 1464.3 (P3)).
Step (c): Formation of Second Cys6-14 Disulfide Bridge (Peptide
1).
The monocyclic precursor from step (b) (72 mg) was dissolved in 75%
AcOH/water (72 mL) under a blanket of nitrogen. 1 M HCl (7.2 mL)
and 0.05 M I.sub.2 in AcOH (4.8 mL) were added in that order and
the mixture stirred for 45 min. 1 M ascorbic acid (1 mL) was added
giving a colourless mixture. Most of the solvents were evaporated
in vacuo and the residue (18 mL) diluted with water/0.1 TFA (4 mL)
and the product purified using preparative HPLC.
Purification by preparative HPLC (gradient: 0% B for 10 min, then
20-30% B over 40 min where A=H.sub.2O/0.1% TFA and B=ACN/0.1% TFA,
flow rate: 10 mL/min, column: Phenomenex Luna 5.mu. C18 (2)
250.times.21.20 mm, detection: UV 214 nm, product retention time:
43-53 min) of the residue afforded 52 mg of pure Peptide 1.
The pure product was analysed by analytical HPLC (gradient: 10-40%
B over 10 min where A=H.sub.2O/0.1% TFA and B=ACN/0.1% TFA, flow
rate: 0.3 mL/min, column: Phenomenex Luna 3.mu. C18 (2) 50.times.2
mm, detection: UV 214 nm, product retention time: 6.54 min).
Further product characterisation was carried out using electrospray
mass spectrometry (MH.sub.2.sup.2+ calculated: 1391.5,
MH.sub.2.sup.2+ found: 1392.5).
Example 2
Synthesis of Compound 1
(Boc-aminooxy)acetic acid (Sigma-Aldrich; 138 mg, 0.72 mmol), EDC
(138 mg, 0.72 mmol) and N-hydroxysuccinimide (83 mg, 0.72 mmol)
were dissolved in DMF (1 ml). The solution was shaken for 25 min,
and then added to a solution of Peptide 1 (1.0 g, 0.36 mmol) in DMF
(5 ml). The reaction mixture was stirred for 2 min. Sym.-collidine
(239 .mu.L, 1.80 mmol) was then added, and the reaction mixture
stirred for 3 hours. The reaction mixture was diluted with water (5
ml), and the product purified by preparative RP-HPLC.
HPLC conditions: Waters Prep 4000 system, Solvent A=H.sub.2O/0.1%
TFA and Solvent B=ACN/0.1% TFA; gradient 20-40% B over 60 min; flow
rate=50 ml/min; column: Phenomenex Luna 10 .mu.m C18 (2)
250.times.50 mm; detection: uv 214 nm.
Yield of purified Compound 1690 mg (65%). Found m/z: 1478.4,
expected MH.sub.2.sup.2+: 1478.1.
Example 3
Synthesis of Compound 3A
Step (a): Preparation of N-(4-fluorobenzylidene)aminooxyacetic
acid.
(Boc-aminooxy)acetic acid (96 mg, 0.50 mmol) and
4-fluorobenzaldehyde (53 .mu.L, 0.50 mmol) were dissolved in formic
acid (0.5 ml), and the reaction mixture stirred for 135 mins. The
reaction mixture was then diluted with 20% ACN/water/0.1% TFA (7
ml), and the product purified by semi-preparative RP-HPLC.
HPLC conditions: Beckman System Gold; Solvent A=H.sub.2O/0.1% TFA
and Solvent B=ACN/0.1% TFA; gradient 25-35% B over 40 min; flow
rate=10 ml/min; column: Phenomenex Luna 5 .mu.m C18 (2)
250.times.21.2 mm; detection: uv 214 nm.
Yield 92 mg (93%).
Step (b): Preparation of Compound 3A
N-(4-Fluorobenzylidene)aminooxyacetic acid [from Step (a), 43 mg,
0.22 mmol] and PyBOP (112 mg, 0.22 mmol) were dissolved in DMF (2
ml). A solution of DIPEA (157 .mu.L, 0.90 mmol) in DMF (10 ml) was
added, and the mixture shaken for 1 min. The solution was then
added to a solution of Peptide 1 (500 mg, 0.18 mmol) in DMF (10
ml), and the reaction mixture shaken for 30 min. The reaction
mixture was then diluted with water (20 ml), and the product
purified by preparative HPLC.
HPLC conditions as per Example 2, except: Solvent A=H.sub.2O/0.1%
ammonium acetate and Solvent B=ACN. Yield 291 mg (55%) of pure
material. Found m/z: 988.6, expected MH.sub.3.sup.3+: 987.7.
Example 4
Synthesis of Compound 3B from Compound 1
Step (a): Deprotection of Compound 1 to Give Compound 2.
Compound 1 (7 mg, 2.37 .mu.M) in a 5-ml reaction vial was treated
with water (10 .mu.L) and trifluoroacetic acid (190 .mu.L), and
then immersed within a sealed vial in a sonic bath for 10 minutes.
The aqueous TFA was then removed in vacuo (approximately 30 mins),
and the residue reconstituted in citrate buffer (pH 2.6, 1.7 mL)
and loaded onto an automated synthesizer cassette (FASTLAB.TM.
chemistry synthesizer platform, GE Healthcare Ltd) at position
14.
Step (b) Synthesis and Purification of .sup.18F-Benzaldehyde.
[.sup.18F]fluoride was produced using a GEMS PETtrace cyclotron
with a silver target via the [.sup.18O](p,n) [.sup.18F] nuclear
reaction. Total target volumes of 1.5-3.5 mL were used. The
radiofluoride was trapped on a Waters QMA cartridge
(pre-conditioned with carbonate), and the fluoride is eluted with a
solution of Kryptofix.sub.2.2.2. (4 mg, 10.7 .mu.M) and potassium
carbonate (0.56 mg, 4.1 .mu.M) in water (80 .mu.L) and acetonitrile
(320 .mu.L). Nitrogen was used to drive the solution off the QMA
cartridge to the reaction vessel. The [.sup.18F]fluoride was dried
for 9 minutes at 120.degree. C. under a steady stream of nitrogen
and vacuum. Trimethylammonium benzaldehyde triflate, [Haka et al,
J. Lab. Comp. Radiopharm., 27, 823-833 (1989)] (3.3 mg, 10.5
.mu.M), in dimethylsulfoxide (1.1 mL) was added to the dried
[.sup.18F]fluoride, and the mixture heated at 105.degree. C. for 7
minutes to produce 4-[.sup.18F]fluorobenzaldehyde. The labelling
efficiency was 69.+-.3% decay corrected.
The crude labelling mixture was then diluted with ammonium
hydroxide solution and loaded onto an MCX+ SPE cartridge
(pre-conditioned with water as part of the FASTLAB.TM. chemistry
synthesizer platform sequence). The cartridge was washed with
water, dried with nitrogen gas before elution of
4-[.sup.18F]fluorobenzaldehyde back to the reaction vessel in
ethanol (1 mL). Approximately 13% (decay corrected) of
[.sup.18F]fluorobenzaldehyde remained trapped on the cartridge.
Step (c): Aldehyde Condensation with Amino-Oxy Derivative (Compound
2).
Compound 2 (5 mg, 1.8 .mu.mol) was transferred to the FASTLAB.TM.
chemistry synthesizer platform reaction vessel prior to elution of
4-[.sup.18F]fluorobenzaldehyde is returned from the MCX+ cartridge.
The mixture was then heated at 70.degree. C. for 17 minutes).
Analytical HPLC confirmed that the RCP of the Compound 3B product
was 63.+-.9%.
The crude reaction mixture was diluted with water (10 mL) and
loaded onto preparative HPLC. A 10 mM ammonium acetate vs
acetonitrile system gave complete separation between the 3 possible
radioactive components of the crude reaction mixture, namely
[.sup.18F]fluoride (T.sub.R=0.5 mins), [.sup.18F]Compound 3B
(T.sub.R=6 mins) and 4-[.sup.18F]fluorobenzaldehyde (T.sub.R=9
mins). Recovery of radioactivity from the HPLC system was good,
with a recovery efficiency of 97%. The purified product was
obtained by collecting the around 6 mins retention time.
Example 5
HPLC Separation of .sup.18F-Labelled c-Met Cyclic Peptide from
Unlabelled Peptide
Compound 3A was prepared according to Example 3.
(i) Analytical HPLC Conditions.
TABLE-US-00007 Column: XBridge Shield RP 18 (4.6 .times. 50) mm,
2.5 .mu.m, Aqueous mobile phase A: 10 mM NH.sub.4Ac (buffer) pH ca.
6.8; Organic mobile phase B: Acetonitrile. Column temperature:
25.degree. C. Flow: 1.2 ml/min. Gradient: Minutes 0 1 16 19 22 22.1
26 % B 20 20 40 100 100 20 20
(ii) Preparative HPLC Conditions
TABLE-US-00008 Column: XBridge Shield RP 18 (10 .times. 100) mm, 5
.mu.m. Aqueous mobile phase A: 10 mM NH.sub.4Ac (buffer) pH ca.
6.8; Organic mobile phase B: Ethanol (90%) Mobile phase A (10%) .
Column temperature: 25.degree. C. Flow: 4 ml/min. Gradient: Minutes
0 1 16 20 25 26 % B 15 15 40 100 100 15
(iii) Analytical and Preparative HPLC Results.
TABLE-US-00009 Analytical HPLC PreparativeHPLC Retention time
Retention time Compound (minutes) (minutes) aniline hydrochloride
1.8 3 fluorobenzaldehyde 4.3 13 Compound 2 4.8 undefined Peptide 1
5.1 undefined Compound 3A 8.8 19
Example 6
Biodistribution of .sup.18F-Labelled c-Met Peptide (Compound 3B) in
Tumour-Bearing Nude Mice
CD-1 male nude mice (ca. 20 g) were housed in individual ventilated
cages, with ad libitum access to food and water. HT-29 cells (ATCC,
Cat. no. HTB-38) were grown in McCoy's 5a medium (Sigma # M8403)
supplemented with 10% fetal bovine serum and
penicillin/streptomycin. Cells were split 1:3 two times a week, at
70-80% confluent using 0.25% trypsin and incubated in 5% CO.sub.2
at 37.degree. C. The mice were injected s.c under light gas
anaesthesia (Isoflurane) with the HT-29 cell suspension at one site
(nape of the neck) with a nominal dose of 10.sup.6 cells per
injections in a volume of 100 .mu.l using a fine bore needle (25
G). The tumours were then allowed to develop for 20 days, or until
at least 200 mm.sup.3 in volume (for inclusion in the study).
After the 20 day growth time, animals were injected with Compound
3B (0.1 ml, 1-5 MBq/animal) as an intravenous bolus via the tail
vein. At various times post injection animals were euthanised,
dissected and the following organs and tissues removed:
The tumour uptake was 2.3% id/g at 2 minutes, peaking at 30 minutes
(3.8% id/g) then decreasing over time to 1.9% id/g at 120 mins pi.
The overall retention within the tumour was 83%. There was
reasonably rapid blood clearance over time (initial 2 minute blood
was 9.2% id/g decreasing to 0.81% id/g at 120 mins pi). Key
background tissue (e.g. lungs and liver) followed the blood
clearance profile over time, with uptake at 120 min p.i. of 1.1%
id/g (liver) and 1.56% id/g (lungs).
Example 7
Receptor Blocking Study of Compound 3B in Tumour-Bearing Nude
Mice
The study of Example 6 was repeated with co-injection of 100 and
1000-fold excess of the non-radioactive analogue, Compound 3A
(.about.1.5 .mu.g and 15 .mu.g excess per animal), with animals
dissected at 120 minutes post injection. All animals in this study
had a similar bodyweight (range of 25 to 30 g). The data
demonstrated that a statistically significant reduction (p<0.01)
in tumour uptake of Compound 3B was achieved with 1000-fold excess
unlabelled peptide (HT-29 tumour uptake fell from 1.9 to 1.1% id/g;
a 40% reduction).
Example 8
Primate PET Imaging of Compound 3B
The biodistribution of Compound 3B in three female cynomolgus
monkeys was measured by PET. Two tracer injections were performed
at each occasion: (a) tracer alone (Compound 3B) 3 MBq/kg (base
line study); (b) tracer 9 MBq/kg with co-injection of with 0.15
mg/kg of Compound 3A (blockade study) four hours after baseline
injection.
The tracer was injected as a bolus dose in 1-3 mL followed by 1 mL
saline.
Blood samples (0.2 ml) for radioactivity determination were taken
at intervals out to 210 minutes after administration. In the
dynamic studies regions of interest were drawn in bone, heart,
kidney, lung, liver, and muscle. In the whole-body studies, regions
of interest were drawn in bone, brain, colon, heart, kidney, lung,
liver, muscle, pancreas, small bowel, spleen, and bladder.
Time-activity data were generated expressed as standard uptake
values (SUV).
Specific binding (.about.40%) was observed in rhesus monkey liver
in vitro using frozen section autoradiography. Rhesus monkey muscle
was not observed to have any specific binding. In vivo studies in
cynomolgus monkey showed a rapid uptake in liver which was reduced
by >40% after co-injection of 0.15 mg/kg of Compound 3A. No
specific binding to muscle in vivo was observed.
Example 9
Effect of Cyclodextrin on the Biodistribution of Compound 3B
The biodistribution of Compound 3B and Compound 3B formulated with
the solubiliser hydroxypropyl-.beta.-cyclodextrin (HPCD) were
compared. No significant differences were found.
Example 10
Fully Automated Synthesis of Compound 3B
The synthesis analogous to Example 4 was carried out, using
Compound 2 dissolved up in buffer or aniline solution and loaded
onto the FASTLAB.TM. chemistry synthesizer platform cassette for
immediate use.
The table below summarises the amount of each of the main
radiolabelled constituents, as calculated by radiochemical purity
of analytical HPLC:
TABLE-US-00010 Compound 1 Compound 2 Product precursor precursor T
= 0 mins Compound 3B 63 .+-. 9% 75 .+-. 3%
[.sup.18F]Fluorobenzaldehyde 28 .+-. 15% 20 .+-. 3% T = 60 mins
Compound 3B 76 .+-. 20% 92 .+-. 2% [.sup.18F]Fluorobenzaldehyde 20
.+-. 15% 2 .+-. 1%
Example 11
Automated Synthesis of Compound 3B Using SPE Purification
The synthesis of Example 4 was carried out using a FastLab.TM.: (GE
Healthcare Ltd) automated synthesiser apparatus. The cassette was
configured with reagents, syringes and SPE cartridges as shown in
FIG. 1.
The QMA (quaternary methyl ammonium water treatment), MCX+ (mixed
cation exchange) and C2 (low hydrophobicity) SPE cartridges were
all obtained from Waters.
During the FASTLAB.TM. chemistry synthesizer platform sequence the
cartridges were (in tandem) conditioned with Ethanol. Immediately
prior to use, the cartridges were primed with dilute (0.2%
phosphoric acid). The crude reaction mixture was diluted with 1%
phosphoric acid and loaded onto the SPE. The SPE was washed with
water before the product was eluted in 6 mL water (80% ethanol),
and the radiochemical purity (RCP) analysed by analytical HPLC.
The results, based on the starting amount of .sup.18F-fluoride
used, were as follows:
TABLE-US-00011 Starting Activity End of Synthesis (MBq) Yield (%)
RCP 493 21 >99% 750 25 >99% 1,000 26 >99% 49,000 19 94%
61,000 18 98% 67,400 21 96%
Example 12
Formulation and Freeze-Drying of Precursor
The peptide precursor Compound 2 (2.5 or 5 mg) was mixed with a
formulation buffer (disodium hydrogen phosphate dihydrate and
citric acid monohydrate, pH 2.8) and stirred until a homogenous
suspension was obtained. 13 mm vials from a FASTlab.TM.; and
automated synthesizer apparatus (GE Healthcare Ltd) with
freeze-drying stoppers were each filled with 1 mL suspension. The
vials were then frozen in the freeze-drying unit and subjected to a
4-day freeze-drying cycle.
All vials containing peptide gave satisfying lyophilised cakes. The
lyophilised precursor was found to dissolve much more quickly than
dry-dispensed Compound 2.
Example 13
Effect of pH on Solubility
Compound 3B was prepared on a FASTLAB.TM. synthesizer, and the
product eluent (.about.6 mL) was collected in a vial containing 2
mL of formulation buffer (pABA, citric buffer, pH 7). A cloudy
solution was observed. The solution (8 ml) was easily filtered
through a pre-filter (Pall 25 mm filter with 0.2 .mu.m Supor
membrane with hydrophobic repel stripe, part no. 6124211), giving a
clear solution.
The filtered solution was divided in to 4.times.2 mL samples and
the pH in the 4 samples adjusted with phosphate (solid, so as not
to change volume) as follows:
TABLE-US-00012 Vial 1 pH 7.5 (original solution, no adjustments)
Vial 2 pH 6.1 Vial 3 pH 8.6 Vial 4 pH 9.1
All samples were stored in the dark at ambient temperature. To
evaluate potential aggregation/precipitation, the 4 samples were
followed by static light scattering for 28 days. The following was
observed: pH 6.1 showed visual precipitate at day 10; no indication
of aggregation at pH.gtoreq.7.5 was observed after 28 days.
Example 14
Human Studies
Imaging with Compound 3B was studied in 6 human patients previously
diagnosed with head and neck squamous cell carcinoma. The agent was
well-tolerated (no adverse effects). 5 of the 6 patients had
moderate/high uptake of the tracer, and 1 patient had low (similar
to contralateral side). This is consistent with the literature
reports of 80% of such patients overexpressing c-Met.
SEQUENCE LISTINGS
1
7117PRTArtificialsynthetic peptide 1Cys Xaa Cys Xaa Gly Pro Pro Xaa
Phe Glu Cys Trp Cys Tyr Xaa Xaa 1 5 10 15 Xaa
218PRTArtificialsynthetic peptide 2Ser Cys Xaa Cys Xaa Gly Pro Pro
Xaa Phe Glu Cys Trp Cys Tyr Xaa 1 5 10 15 Xaa Xaa
322PRTArtificialsynthetic peptide 3Ala Gly Ser Cys Xaa Cys Xaa Gly
Pro Pro Xaa Phe Glu Cys Trp Cys 1 5 10 15 Tyr Xaa Xaa Xaa Gly Thr
20 44PRTArtificialsynthetic peptide 4Gly Gly Gly Lys 1
54PRTArtificialsynthetic peptide 5Gly Ser Gly Lys 1
65PRTArtificialsynthetic peptide 6Gly Ser Gly Ser Lys 1 5
726PRTArtificialsynthetic peptide 7Ala Gly Ser Cys Tyr Cys Ser Gly
Pro Pro Arg Phe Glu Cys Trp Cys 1 5 10 15 Tyr Glu Thr Glu Gly Thr
Gly Gly Gly Lys 20 25
* * * * *